Regeneration of both plant tissues and transgenic plant tissues using a new plant hormone, 5-bromoindole-3-acetic acid

ABSTRACT

The present invention describes the use of 5-bromoindole-3-acetic acid (5-B-IAA) as an auxin affecting plant cell growth. The invention relates to the use of 5-B-IAA compositions to affect growth in monocotyledonous as well as in dicotyledonous plants. The invention also describes the use of 5-B-IAA in plant growth affecting compositions for the regeneration of both plant tissues and transgenic plant tissues. Further, the invention provides plant growth affecting compositions comprising 5-B-IAA alone or in a mixture comprising one or more additional plant growth regulators, such as cytokinin, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/392,177, filed Sep. 9, 1999 (now U.S. Pat. No. 6,271,032 issued Aug.7, 2001), which is a continuation of U.S. patent application Ser. No.08/861,666, filed May 22, 1997 (now U.S. Pat. No. 5,994,135 issued Nov.30, 1999), which is a continuation of U.S. patent application Ser. No.08/430,209 filed Apr. 27, 1995 (now U.S. Pat. No. 5,674,731 issued Oct.7, 1997), all of which are incorporated by reference in their entiretyherein.

FIELD OF THE INVENTION

This invention relates to the use of 5-bromoindole-3-acetic acid(5-B-IAA) as a plant hormone stimulatory to cell growth. It particularlyrelates to the use of 5-B-IAA in plant growth affecting compositions forthe regeneration of both plant tissues and transgenic plant tissues.

BACKGROUND OF THE INVENTION

Plant growth is affected by a variety of physical and chemical factors.Physical factors include available light, day length, moisture andtemperature. Chemical factors include minerals, nitrates, cofactors,nutrient substances and plant growth regulators or hormones, forexample, auxins, cytokinins and gibberellins.

Indole-3-acetic acid (IAA) is a naturally-occurring plant growth hormoneidentified in plants. IAA has been shown to be directly responsible forincrease in growth in plants in vivo and in vitro. The characteristicsinfluenced by IAA include cell elongation, internodal distance (height),leaf surface area and crop yield. IAA and other compounds exhibitinghormonal regulatory activity similar to that of IAA are included in aclass of plant regulators called “auxins.”

Compounds known to function as auxins in plants include, for example,4-chloroindole-3-acetic acid (4-Cl-IAA) which is a naturally occurringplant growth regulator, acting to induce stem elongation and to promoteroot formation. Whereas IAA is found in most organs of a plant, 4-Cl-IAAwas shown to be present in immature and mature seeds of Pisum sativum,but not in any other organ (Ulvskov et al. (1992) 188:182-189). Somesynthetic auxins include naphthalene-1-acetic acid (NAA),5,6-dichloro-indole-3-acetic acid (5,6-Cl₂-IAA),4-chloro-2-methylphenoxyacetic acid (MCPA); 2,4-chlorophenoxyacetic acid(2,4D); 2,4,5-trichlorophenoxyacetic acid (2,4,5-T);2-(4-chloro-2-methylphenoxy) propionic acid (CMPP);4-(2,4-dichlorophenoxy) butyric acid (2,4-DB); 2,4,5-trichlorobenzoicacid (TBA); and 3,5-dichloro-2-methoxybenzoic acid (dicamba), forexample. All the above acids are active in the form of their salts andesters, such as their sodium, potassium, ammonium, dimethylamine andethanolamine salts, and their lower alkyl esters. Many of thesesynthetic auxins are being used commercially as effective herbicides andsome of them are known to adversely affect morphogenesis of treatedplants.

Preparations based on cytokinins, such as 6-furfurylamino purine(kinetin) and 6-benzylamino purine (BAP), are also known to be growthstimulators. However, cytokinin-based preparations are most effective incombination with auxins. While the mechanism by which cytokinins affectthe growth cycle of plants is far from being understood, it is apparentthat they affect leaf growth and prevent aging in certain plants.

It is a general objective in the field to successfully regenerate plantsof major crop varieties using methods such as tissue culture and geneticengineering. The art of plant tissue culture has been an area of activeresearch for many years but over the past five to ten years anintensified scientific effort has been made to develop regenerable planttissue culture procedures for the important agricultural crops such asmaize, wheat, rice, soybeans, and cotton.

In vitro culture techniques are well established in plant breeding(Reinert, J., and Bajaj, Y. P. S., eds. (1977) Plant Cell, Tissue andOrgan Culture, Berlin: Springer; Simmonds, N. W. (1979) Principles ofCrop Improvement, London: Longman; Vasil, I. K., Ahuja, M. K. and Vasil,V. (1979) “Plant tissue cultures in genetics and plant breeding,” Adv.Genet. 20:127-215). First, embryo culture has, for decades, been avaluable adjunct to making difficult interspecific crosses. Second, morerecent but also well established, is shoot-tip culture, which finds usesin rapid clonal multiplication, development of virus-free clones andgenetic resource conservation work. Both techniques depend upon theretention of organizational integrity of the meristem. A step furthertakes us to callus, cell, and protoplast cultures in which organizationis lost but can in most cases be recovered. A step further still takesus to in vitro hybridization, which has, after regeneration, yieldedinterspecific amphidiploids. The technique may provide desiredamphidiploids which cannot be made by conventional means, and presentspossibilities for somatic recombination by some variant of it. Theforegoing techniques are widely in use (Chaleff, R. S. (1981) Geneticsof Higher Plants, Applications of Cell Culture, Cambridge: CambridgeUniversity Press).

Plant genetic engineering techniques enable the following steps: (a)identification of a specific gene; (b) isolation and cloning of thegene; (c) transfer of the gene to recipient plant host cells: (d)integration, transcription and translation of the DNA in the recipientcells; and (e) multiplication and use of the transgenic plant (T.Kosuge, C. P. Meredith and A. Hollaender, eds (1983) Genetic Engineeringof Plants, 26:5-25; Rogers et al. (1988) Methods for Plant MolecularBiology [A. Weissbach and H. Weissbach, eds.] Academic Press, Inc., SanDiego, Calif.). Newly inserted foreign genes have been shown to bestably maintained during plant regeneration and are transmitted toprogeny as typical Mendelian traits (Horsch et al. (1984) Science223:496, and DeBlock et al. (1984) EMBO 3:1681). The foreign genesretain their normal tissue specific and developmental expressionpatterns.

Successful transformation and regeneration techniques have beendemonstrated in the prior art for many plant species. The Agrobacteriumtumefaciens-mediated transformation system has proved to be efficientfor many dicotyledonous plant species. For example, Barton et al. (1983,Cell 32:1033) reported the transformation and regeneration of tobaccoplants, and Chang et al. (1994, Planta 5:551-558) described stablegenetic transformation of Arabidopsis thaliana.

The Agrobacterium method for gene transfer was also applied tomonocotyledonous plants, e.g.,in plants in the Liliaceae andAmaryllidaceae families (Hooykaas-Van Slogteren et al., 1984, Nature311:763-764) and in Dioscorea bulbifera (yam) (Schafer et al., 1987,Nature 327:529-532); however, this method did not appear to be efficientfor the transformation of graminaceous monocots, which include such foodcrops as wheat, rice and corn.

Transformation of food crops was obtained with alternative methods,e.g., by polyethylene glycol (PEG)-facilitated DNA uptake (Uchimiya etal. (1986) Mol. Gen. Genet. 204:204-207) and electroporation (Fromm etal. (1986) Nature 319:791-793), both of which used protoplasts astransfer targets. Monocot and dicot tissues may be transformed bybombardment of tissues by DNA-coated particles (Wang et al. (1988) PlantMol. Biol. 11:433-439; Wu, in Plant Biotechnology (1989), Kung andArntzen, Eds., Butterworth Publishers, Stoneham, Mass.). Regenerationwas described in rice (Abdullah et al. (1986) Bio/Technology4:1087-1090) and maize (Rhodes et al. (1988) Bio/Technology 6:56-60 and(1988) Science 240:204-207).

SUMMARY OF THE INVENTION

The principal object of the present invention is to provide a growthaffecting composition comprising 5-bromoindole-3-acetic acid (5-B-IAA),or ester or salt derivatives of 5-B-IAA, in order to achieve a plantgrowth affecting response. The invention contemplates the use of 5-B-IAAto affect growth in both monocotyledonous and dicotyledonous plants.

It is also an object of the invention to provide a composition foraffecting plant growth comprising a mixture of 5-B-IAA and one or moreadditional plant growth regulators, for example, a cytokinin, agibberellin, etc., in definite proportions for wide application tovarious plants in order to achieve a plant growth affecting response. Inspecific embodiments, the invention was exemplified with compositionscomprising 5-B-IAA and a cytokinin to affect the growth of plants.

It is a further object of the invention to provide a composition foraffecting plant growth comprising 5-B-IAA or a mixture of 5-B-IAA andcytokinin as a component of media which sustain the plant during plantdevelopment or tissue regeneration and also serve as a vehicle whereby a5-B-IAA-comprising composition may be applied.

It is an additional object of the invention to provide a method ofaffecting plant growth which comprises the step of applying an effectiveamount of the plant growth affecting composition comprising 5-B-IAA or amixture of 5-B-IAA and one or more additional plant growth regulators,for example, a cytokinin, a gibberellin, etc., to a plant species, aplant locus, cell or tissue, or a seed of a plant. In specificembodiments, the invention was exemplified with compositions comprising5-B-IAA and a cytokinin to affect the growth of plants.

Furthermore, it is an object of the invention to provide a method ofaffecting plant growth which comprises the step of applying an effectiveamount of the plant growth affecting composition comprising 5-B-IAA or amixture of 5-B-IAA and one or more additional plant growth regulators,for example, a cytokinin, a gibberellin, etc., to a transgenic plant. Inspecific embodiments, the invention was exemplified with compositionscomprising 5-B-IAA and a cytokinin to affect the growth of plants.

DESCRIPTION OF THE FIGURES

FIG. 1 documents plant growth stimulated by 5-B-IAA or IAA in thepresence of different concentrations of BAP on the formation of root,shoots and calli. New Auxin=5-B-IAA.

FIG. 2 presents the regeneration of embryogenic callus from mature seedsof Oryza sativa cv. Orion stimulated in a first medium comprising 2,4-D(left) and in a second medium comprising 5-B-IAA and BAP (right).

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are provided in order to provide clarity as tothe intent or scope of their usage in the specification and claims.

The term 5-bromoindole-3-acetic acid or 5-B-IAA as used herein refersnot only to the free acid form but also to an amide, an ester or a saltform of 5-B-IAA. Included in the meaning of 5-B-IAA are, for example,such salt and ester derivatives as the sodium, potassium, ammonium,dimethylamine, ethanolamine, etc. salts and amides and the lower alkylesters.

The term plant growth regulator or hormone as used herein refers to anaturally occurring or synthetic compound that acts as a hormone inregulating plant growth. Important growth regulators are exemplified byauxins, cytokinins and gibberellins.

The term auxin or cytokinin as used herein refers to a plant growthregulator that affects the growth of plants. An auxin is exemplified bya compound such as indole-3-acetic acid (IAA), indole-3-butyric acid(IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid(NAA), 5,6-dichloroindole-3-acetic acid (5,6-Cl₂-IAA) and the like. Acytokinin is exemplified by a compound such as 6-benzylamino purine(BAP), N⁶.(Δ₂ isopentenyl) adenine (2iP), isopentenylpyrophosphate(ipp), 6-(4-hydroxy-3-methyl-2-transbetenylamino)purine (zeatin),6-furfurylaminopurine (kinetin) and the like. A compound can be testedfor auxin activity using a bioassay, e.g., the elongation of coleoptilesof Avena sativa (Bottger et al. (1978) Planta 140:89) or the root growthinhibition of Chinese cabbage (Marumo et al. (1974) in Plant GrowthSubstance, p. 419, Hirokawa Publishing Co., Inc., Tokyo) or thehypocotyl swelling of mung bean (Marumo et al. (1974) supra). Cytokininactivity may be measured in assays designed to evaluate the promotion ofgrowth in plants (e.g., tobacco bioassays, etc.) as is well known in theart (Skoog et al. 1967) Phytochem 6:1169-1192; Morris (1986) Ann. Rev.Plant Physiol. 37:509-538; Horgan (1984) in Advanced Plant Physiol(Wilkins, M. B., ed.) pp. 53-75, Pitman Publishing, London; Letham andPalni (1983) Ann. Rev. Plant Physiol 34:163-197; and Chen (1981) inMetabolism and Molecular Activities of Cytokinins (Guern, J. andPeaud-Lenoel, C., eds., Springer, New York, pp. 34-43). Variations ofthe cytokinin/auxin concentration ratio cause the enhancement in plantgrowth to occur preferentially in certain tissues. For example, a highcytokinin/auxin ratio promotes growth of shoots, whereas a low cytokininto auxin ratio promotes the growth of roots (Depicker et al. (1983) inGenetic Engineering of Plants, T. Kosunge, C. P. Meredith and A.Hollaender, eds., Plenum Press, New York, p. 154).

The term a plant as used herein refers to a whole plant or a part of aplant comprising, for example, a locus of a plant, a cell of a plant, atissue of a plant, an explant, or seeds of a plant. This term furthercontemplates a plant in the form of a suspension culture or a tissueculture including, but not limited to, a culture of calli, protoplasts,embryos, organs, organelles, etc.

The term transformed plant or transformed plant tissues as used hereinrefers to introduction of a foreign DNA into a plant or plant tissue andexpression of the DNA in the plant or plant tissue.

The term transgenic plant or transgenic plant tissue as used hereinrefers to a plant or plant tissue stably transformed with a foreign geneintroduced into the genome of the individual plant cells.

The term transient expression refers to a plant or plant tissuetransformed with a DNA, where that DNA is expressed only for a shortperiod of time immediately after transformation.

The term genetic engineering as used herein refers to the introductionof foreign, often chimeric, genes into one or more plant cells which canbe regenerated into whole, sexually competent, viable plants which canbe self-pollinated or cross-pollinated with other plants of the samespecies so that the foreign gene, carried in the germ line, can beinserted into or bred into agriculturally useful plant varieties.

The term regeneration as used herein refers to the production of atleast one newly developed or regenerated plant tissue, e.g., root,shoot, callus, etc., from a cultured plant tissue or unit, e.g., leafdisc, seed, etc.

The terms percent regeneration, % regeneration or regenerationefficiency as used herein refer to the number of tissue cultured plantunits producing at least one newly developed or regenerated tissue as apercentage of the total number of tissue cultured plant units, e.g.,

(number of leaf discs with shoots/total number of leaf discs×100).

The terms affecting plant growth or growth affecting or affector oraffect as used herein refer to any one of a number of plant responseswhich improve or change, relative to what is observed in the absence ofthe growth regulator, some characteristic of overall plant growth, forexample, stimulation of seed germination, inducing rooting, suppressingshooting, promoting cell proliferation, stimulating callus growth, etc.

The term effective amount as used herein refers to the amount orconcentration of a compound that is a plant growth regulator or hormoneadministered to a plant such that the compound stimulates or invokes oneor more of a variety of plant growth responses. A plant growth responseincludes, among others, the induction of stem elongation, the promotionof root formation, the stimulation of callus formation, enhancement ofleaf growth, stimulation of seed germination, increase in the dry weightcontent of a number of plants and plant parts, and the like.

The present invention relates to the discovery that 5-B-IAA, achemically synthesized compound that is commercially available, hasutility as an auxin in affecting plant growth. (Of eleven commerciallyavailable IAA analogs tested for tobacco plant growth regulatoryactivity, only 5-B-IAA exhibited the ability to stimulate root andcallus formation. In combining 5-B-IAA with cytokinin, both callus andshoot formation were observed.

5-B-IAA was found to be superior to IAA in functioning as an auxin inboth monocots and dicots. For example, 5-B-IAA was between two and fourtimes more effective than IAA in stimulating the regeneration of greencalli from Arabidopsis thaliana. The effect of 5-B-IAA is all the moreremarkable in light of the prior art teaching for Arabidopsis tissueculture responses that “callus induction and regeneration frequenciesare high for root, lower for anther and stem and lowest for leafexplants.” In accordance with the present invention, 5-B-IAA gave anefficiency of 100% for regeneration from Arabidopsis thaliana leaves.

Superiority of 5-B-IAA was also observed in monocot regeneration. Priorart methods used to obtain tissue regeneration from monocotyledonousplants, for example, rice, require approximately three months andincubation of immature seeds in two different culture media. Incontrast, in accordance with the present invention using 5-B-IAA asauxin, regeneration of shoots from rice embryonic callus derived frommature seeds was obtained in about one and a half months, requiring onlyone incubation medium comprising 5-B-IAA and a cytokinin (e.g., BAP) andyielding a regeneration efficiency of 100%. In all bioassays performedto show regeneration from plant tissue and from transgenic plant tissue,5-B-IAA functioned as an auxin to stimulate growth at least as well as,and in many cases better than, IAA, the auxin standard of the art.

Growth affecting compositions of the present invention comprise 5-B-IAA,or a mixture of 5-B-IAA and one or more additional plant growthregulators, such as cytokinin, gibberellin or the like, mixed with acarrier or auxiliary nutrients. The use of BAP, 2iP and kinetin has beenexemplified in particular embodiments of this invention. It iscontemplated that other cytokinins or other plant growth regulatorsknown to the art can be utilized with 5-B-IAA to make a growth affectingcomposition of the invention. It is also contemplated that more than onecytokinin or a different plant growth regulator (e.g., gibberellin,etc.) can be admixed with 5-B-IAA to make a growth enhancing compositionof the invention. Also, the choice of plant growth regulator can bevaried at different stages of the incubation or application cyclescharacterizing the growth of a particular plant. Plant growth regulatorsare known to the art and include, but are not limited to, BAP, 2iP, ipp,zeatin, kinetin, gibberellin, and the like, as described in Skoog et al.(1967) Phytochemistry 6:1169-1192 and Theologis (1989) in PlantBiotechnology (Kung and Arntzen, eds.) Butterworth Publishers, Stoneham,Mass.

The mechanism by which compositions comprising 5-B-IAA of the presentinvention effect the growth cycle of plants and plant tissues is notfully understood at present but it is apparent, as will be demonstratedhereinafter, that they play a significant role in inducing a number ofgrowth affecting responses in a variety of plant species.

The practice of the present invention contemplates a wide variety ofplant growth responses, including stimulation of seed germination andbreaking of dormancy; increasing yields; hastening ripening and colorproduction in fruit; increasing flowering and fruiting; stimulatingshoot formation; inducing callus development; inducing rooting andcausing cell proliferation; increasing the hardiness of various plantspecies; and increasing the dry weight content of a number of plants andplant parts. In addition to these categories of responses, any othermodification of a plant, seed, fruit or vegetable, so long as the netresult is to increase the growth or maximize any beneficial or desiredproperty of the agricultural and horticultural crop or seed, is intendedto be included within the scope of advantageous responses achieved bythe practice of the present invention.

Suitable applications of the growth enhancing compositions of thepresent invention to cultures of plant tissues were shown to induce theregeneration of shoots, roots or calli. This effect was exemplified inboth monocotyledonous and dicotyledonous plant species and is applicableto a wide variety of plants.

The compositions of the instant invention were further utilized forplant regeneration from transgenic plants.

Genetic engineering of plants generally involves two complementaryprocesses. The first process involves the genetic transformation of oneor more plant cells of a specifically characterized type. Bytransformation it is meant that a foreign gene, typically a chimericgene construct, is introduced into the genome of the individual plantcells, typically through the aid of a vector which has the ability totransfer the gene of interest into the genome of the plant cells inculture. The second process then involves the regeneration of thetransformed plant cells into whole sexually competent plants. Neitherthe transformation nor regeneration process need be 100% successful butmust have a reasonable degree of reliability and reproducibility so thata reasonable percentage of the cells can be transformed and regeneratedinto whole plants.

The two processes, transformation and regeneration, must becomplementary. The complementarity of the two processes must be suchthat the tissues which are successfully genetically transformed by thetransformation process must be of a type and character, and must be insufficient health, competency and vitality, so that they can besuccessfully regenerated into whole plants.

Successful transformation and regeneration techniques have beendemonstrated for monocots and dicots in the prior art. For example, thetransformation and regeneration of tobacco plants was reported in Bartonet al., Cell 32:1033 (April 1983), whereas the regeneration of cotton isdescribed in Umbeck, U.S. Pat. No. 5,004,863, issued Apr. 2, 1991.Further, transformation and regeneration of rice was described byAbdullah et al. (1986) Bio/Technology 4:1087-1090, whereas maize wastransformed and regenerated as described in Rhodes et al. (1988)Bio/Technology 6:56-60 and Science 240:204-207.

The most common methodology used for the transformation of cells ofdicot plant species involves the use of the plant pathogen Agrobacteriumtumefaciens. Although Agrobacterium-mediated transformation has beenachieved in some monocots, other methods of gene transfer have been moreeffective, e.g., the polyethylene glycol method, electroporation, directinjection, particle bombardment, etc., as described by Wu in PlantBiotechnology (1989) pp. 35-51, Butterworth Publishers, Stoneham, Mass.The present invention will be useful with any method of transformationthat includes plant regeneration steps.

In a specific embodiment, the invention envisions the genetictransformation of tissues in culture derived from leaf discs orhypocotyl explants. The transformed tissues can be induced to form planttissue structures, which can be regenerated into whole plants.

The transformation technique of the present invention is one which makesuse of the Ti plasmid of A. tumefaciens. In using an A. tumefaciensculture as a transformation vehicle, it is most advantageous to use anon-oncogenic strain of the Agrobacterium as the vector carrier so thatnormal non-oncogenic differentiation of the transformed tissue ispossible. To be effective once introduced into plant cells, the chimericconstruction including a foreign gene of interest must contain apromoter which is effective in plant cells to cause transcription of thegene of interest and a polyadenylation sequence or transcription controlsequence also recognized in plant cells. Promoters known to be effectivein plant cells include the nopaline synthase promoter, isolated from theT-DNA of Agrobacterium, and the cauliflower mosaic virus 35S promoter.Other suitable promoters are known in the art. It is also preferred thatthe vector which harbors the foreign gene of interest also containtherein one or more selectable marker genes so that the transformedcells can be selected from non- transformed cells in culture. In manyapplications, preferred marker genes include antibiotic resistance genesso that the appropriate antibiotic can be used to segregate and selectfor transformed cells from among cells which are not transformed.

The details of the construction of the vectors containing such foreigngenes of interest are known to those skilled in the art of plant geneticengineering and do not differ in kind from those practices which havepreviously been demonstrated to be effective in tobacco, petunia andother model plant species. The foreign gene should obviously be selectedas a marker gene (Jefferson et al. (1987) EMBO J. 6:3901-3907) or toaccomplish some desirable effect in plant cells. This effect may begrowth promotion, disease resistance, a change in plant morphology orplant product quality, or any other change which can be accomplished bygenetic manipulation. The chimeric gene construction can code for theexpression of one or more exogenous proteins, or can cause thetranscription of negative strand RNAs to control or inhibit either adisease process or an undesirable endogenous plant function.

To initiate the transformation and regeneration process for planttissues, it is necessary to first surface sterilize tissues to preventinadvertent contamination of the resulting culture. If the tissues areseeds, the seeds are then allowed to germinate on an appropriategerminating medium containing a fungicide. Four to ten days aftergermination the hypocotyl portion of the immature plant is removed andsectioned into small segments averaging approximately 0.5 centimetersapiece. The hypocotyl explants are allowed to stabilize and remainviable in a liquid or agar plant tissue culture medium.

Once the tissues have stabilized, they can promptly be inoculated with asuspension culture of transformation competent non-oncogenicAgrobacterium. The inoculation process is allowed to proceed for a shortperiod, e.g., two days, at room temperature, i.e., 24° C.

At the end of the inoculation time period, the remaining treated tissuescan be transferred to a selective agar medium, which contains one ormore antibiotics toxic to Agrobacterium but not to plant tissues, at aconcentration sufficient to kill any Agrobacterium remaining in theculture. Suitable antibiotics for use in such a medium includecarbenicillin cefotaxime, etc. as the bacteriocide for Agrobacterium andkanamycin as the selective antibiotic for transformed plant tissues.

The tissues are now cultivated on a tissue culture medium which, inaddition to its normal components, contains a selection agent. Theselection agent, exemplified herein by kanamycin, is toxic tonon-transformed cells but not to transformed cells which haveincorporated genetic resistance to the selection agent and areexpressing that resistance. A suitable tissue culture medium is the MSmedium to which is added the phytohormones 5-B-IAA and a cytokinin, withor without antibiotics. The surviving transformed tissues aretransferred to a secondary medium to induce tissue regeneration. Thesurviving transformed tissue will thus continue to be regenerated into awhole plant through the regeneration technique of the present inventionor through any other alternative plant regeneration protocols.

The precise amount of growth affecting compositions employed in thepractice of the present invention will depend upon the type of responsedesired, the formulation used and the type of plant treated. Theinvention contemplates the use of a ratio of cytokinin concentration toauxin concentration of between approximately 50.0 and 0.001, andpreferably between approximately 5.0 and 0.05, and more preferablybetween approximately 2.0 and 0.25.

The chemical compounds employed as active components of the growthenhancing compositions of the present invention may be prepared inaccordance with processes well known in the prior art or may be obtainedcommercially from readily available sources.

The present compositions may be applied at any developmental stage ofthe plant species to obtain plant hormone or maintenance effectsthroughout maturity and to expedite regrowth in damaged tissues duringearly developmental stages, depending upon the concentration used, theformulation employed and the type of plant species treated.

The compositions of the present invention are preferably used inconjunction with specific auxiliary nutrients or other plant growthregulators in precise proportions to achieve a particular synergistic,growth enhancing response in various type of plants. The presentcompositions may additionally be used in association with fungicides toincrease the disease resistance of various plants, making the planttissue resistant to invasion by pathogens by influencing the enzyme andplant processes which regulate natural disease immunity. While thepresent compositions possess essentially no phytotoxic activity of theirown, they may sometimes be used in conjunction with herbicides tostimulate the growth of unwanted plants in order to make such plantsmore susceptible to a herbicide. However, it is preferred to regard theresults achieved in the practice of the present invention as growthenhancing responses in agricultural and horticultural crops, as well asperennial and annual household plants species.

The following examples are illustrative of the wide range of plantgrowth responses that can be realized by application of a preferredcomposition of the present invention to various plant species.Nevertheless, there is no intention that the invention be limited tothese optimum ratios of active components since workers in the art willfind the compositions of the invention set forth hereinabove to beeffective growth enhancers. Also, it should readily occur to one skilledin the art that the recognition of improved results using thecompositions according to the present invention in connection with otherplants, seeds, fruits and vegetables not specifically illustrated hereinis readily within the capabilities of one skilled in the art. Thefollowing examples serve to illustrate the utility of the inventionwithout limiting its scope.

EXAMPLES Example 1 Evaluation of Auxin Activity in Tobacco

Seeds of Nicotiana tobaccum Xanthi were provided by Dr. James Saunders(USDA, Beltsville, Md.). Young tobacco leaves were removed and cut intosmall pieces. The explants were incubated on (1) MS complete mediumcontaining different concentrations of auxin related compounds only or(2) MS complete medium containing different concentrations ofauxin-related compounds and 0.5 mg/l benzylaminopurine (BAP) using an 18h light/6 h dark cycle until the formation of green calli and shoots.

The regeneration of plant tissues using tissue culture depends on planthormones such as auxin and cytokinin. It is known that the presence ofan auxin in plant tissue cultured on Murashige and Skoog (MS) medium(Murashige, T. and F. Skoog (1962) Physiol. Plant 15:473-497) stimulatesthe formation of root structure whereas the formation of callus isobserved when not only the auxin but also a cytokinin complement the MSnutrient medium. Therefore, evaluation of a test compound as a potentialnew auxin was performed by incubating tobacco leaf discs in (a) the MScomplete medium containing different concentrations of auxin only and(b) the MS complete medium containing different ratios of cytokinin toauxin concentrations (cytokinin/auxin).

Thirteen commercially available compounds were tested for plant growthregulatory activity. As shown in Table 1, only 5-B-IAA exhibited theability to stimulate root and shoot formation. Different concentrationsof each compound were tested for the ability to stimulate root formationfrom tobacco leaf discs. The ability to stimulate root formation wasevaluated for each compound in the presence of different concentrationratios of cytokinin (BAP) to test compound.

TABLE 1 Evaluation of different test compounds for auxin activity usingtobacco leaf disc. Regeneration from Tobacco Leaf Discs Root FormationCallus Formation Test Ratio of BAP/ Compound (mg/l) Test compound TestCompounds 0.5 1.0 2.0 2.0 1.0 0.5 N_(α)-acetyl-L-histidine — — — — — —monohydrate (+)-6-aminopenicilianic acid — — — — — — DL-α-amino-2-thio-— — — — — — pheneacetic acid 5-chloroindole-2-carboxylic — — — — — —acid 2-furanacrylic acid — — — — — — 5-hydroxyindole-3-acetic acid — — —— — — 5-hydroxy-2-indolecarboxylic — — — — — — acid(±)-3-oxo-1-indancarboxylic — — — — — — acid (R)-(−)-2-phenylglycine — —— — — — (S)-(+)-2-2phenylglycine — — — — — — 5-bromoindole-3-acetic acid+++ +++ +++ +++ +++ +++ +++ indicates stimulatory activity − indicatesno activity

FIG. 1 demonstrates that 5-B-IAA functions as an effective auxin.Tobacco leaf discs grown on MS complete medium containing 5-B-IAA or IAAin the presence of low cytokinin concentrations demonstrated rootformation, whereas tobacco leaf discs grown on MS complete mediumcontaining 5-B-IAA and higher cytokinin (BAP) concentrations exhibitedcallus formation.

Example 2 Evaluation of 5-B-IAA auxin activity in Arabidopsis thaliana

Seeds of Arabidopsis thaliana ecotypes Columbia and Landersberg eretawere provided by Dr. Keith Davis (The Ohio State University, Columbus).Tissues of hypocotyl were removed from 10-day-old seedlings, transferredto MS complete medium containing (1) different concentrations of IAA or5-B-IAA with different concentrations of N⁶. (Δ₂Isopentenyl) adenine(2iP) and (2) different concentrations of 5-B-IAA with differentconcentrations of 2iP, BAP, or kinetin. The explants were incubated at23° C. using an 18 h light/6 h dark cycle until the formation of greencalli and shoots.

5-B-IAA was found to be effective in promoting the regeneration of greencallus from Arabidopsis thaliana Columbia and Landersberg. Tissuecultures of A. thaliana leaves removed from 21-day-old seedlings weregrown on MS medium comprising either IAA or 5B-IAA and the cytokinin,2iP. The concentrations of both auxin and cytokinin were varied as shownin Table 2.

TABLE 2 Effect of 5-bromoindole-3-acetic acid on the regeneration ofgreen callus from Arabidopsis thaliana Columbia leaves Conc. Type %regeneration of green callus of 2iP of Concentration of auxin (mg/l)(mg/l) auxin 0.5 1.0 2.0 4.0 0.5 IAA 25.0 58.3 33.3 25.0 0.5 5-B-IAA50.0 100 83.3 91.6 2.0 IAA 23.0 33.3 25.0 25.0 2.0 5-B-IAA 58.3 83.3 10090.9

As indicated in Table 2, at all concentrations of auxin and cytokinintested, 5-B-IAA was superior to IAA in stimulating the regeneration ofgreen calli. Notably, an 100% regeneration efficiency of green calluswas obtained using 5-B-IAA (see Table 2). In contrast, only lowregeneration efficiencies have been reported (Koncz et al., (1992)Methods in Arabidopus Research, pp. 224-273) for the regeneration ofgreen callus from A. thaliana using auxins such as IAA, 2,4-D or NAA.

Further, the effect of 5-B-IAA on the regeneration of green callus wastested in the presence of other cytokinins. As documented in Table 3, ahigh efficiency of regeneration was observed for 5-B-IAA with each ofthe three cytokinins tested, i.e., kinetin, 2iP and BAP. Moreover, 100%regeneration efficiencies were achieved for 5-B-IAA in the presence ofkinetin as well as 2iP. Ratios of cytokinin to auxin of between 2.0 and0.5 were found to be particularly effective.

TABLE 3 Effect of different cytokinins and 5-bromoindole-3-acetic acidon the regeneration of green callus from A. thalaianas Columbia leaves %regeneration of green callus Type of Ratio of Cytokinin/5-B-IAAcytokinin 2.0 1.0 0.5 Kinetin 90% 100% 100% 2iP 100% 100% 100% BAP 70%90% 80%

Example 3 Evaluation of 5-B-IAA Auxin Activity in Rice

Seeds of Oryza sativa cv. Orion were kindly provided by Dr. JamesSaunders (USDA, Beltsville, Md.). For rice embryogenic callus formation,the surfaces of the seeds were sterilized as follows: mature seeds weresoaked in 0.5% detergent with shaking for 1 h, transferred to a solutioncontaining 20% bleach and 0.1% Tween20® and vacuumed with shaking. Theseeds were then rinsed with sterilized distilled water three times. Atthis point the seeds were transferred onto a MS complete mediumcontaining different concentrations of BAP and different concentrationsof 5-B-IAA, incubated in the dark at 25° C. for 1 month, and thenincubated at 25° C. using an 18 h light/6 h dark cycle until theformation of green calli and shoots.

5-B-IAA was found to be effective on the regeneration of embryogeniccallus from seeds of a monocotyledonous plant, e.g., rice. Tissueregeneration from monocotyledons is known to be a challenge with respectto regeneration efficiency as well as incubation time. For example, inrice, in order to obtain regenerated shoots from embryogenic calli, ittakes approximately three months and requires incubation of cultures intwo different media (MS complete medium containing 2,4-D and MS completemedium containing IAA and BAP).

In accordance with this invention, washed seeds of Oryza sativa cv.Orion were incubated on MS complete medium comprising BAP and differentconcentrations of 5-B-IAA. As shown in Table 4,

TABLE 4 Effect of 5-bromoindole-3-acetic acid on the regeneration ofembryogenic green callus from seeds of Oryza sativa cv. Orion. Conc. of% regeneration of embryogenic callus BAP Concentration of 5-B-IAA(mg/l)(mg/l) 1.0 2.0 4.0 8.0 0.5 — 33% 100% 90% 1.0 28% 55% 100% — 2.0 12% 57%55% —

shoot regeneration was obtained with 5-B-IAA at concentrations ofapproximately between 2.0 and 8.0 mg/l in the presence of BAP atconcentrations of approximately between 0.5 and 2.0 mg/l. Notably, shootregeneration from embryogenic callus was observed after onlyapproximately one and a half months in culture and upon incubation inonly one nutrient medium, i.e., an MS complete medium containing 5-B-IAAand BAP. Moreover, under these conditions with 5-B-IAA, an efficiency of100% was achieved for shoot regeneration from embryogenic callus of amonocotyledonous plant as shown in Table 4.

Example 4 The use of 5-B-IAA for Stimulating the Regeneration ofTransgenic Plants

a) Tobacco

Plant transformation was carried out according to theAgrobacterium-mediated transformation procedure essentially as describedby Lin et al. [(1994) Focus 16:72-77)].

For tobacco, the leaf discs were incubated with 10¹⁰ cells/mlAgrobacterium tumefaciens LBA4404 cells, containing pBI121 harboring theGUS reporter gene, in MS complete medium with 0.5 mg/l2-(N-morpholino)ethanesulfonic acid (MES) for 10 min, transferred tosolid MS complete medium, and incubated for 2 days at 25° C., using an18 h light/6 h dark cycle for cocultivation. After cocultivation, theexplants were transferred to MS media containing different ratios ofBAP/IAA or BAP/5-B-IAA, 100 mg/l kanamycin and 500 mg/l carbenicillinand incubated at 25° C. using an 18 h light/6 h dark cycle for shootformation.

5-B-IAA was found to be effective in stimulating the regeneration ofshoots from to transgenic tobacco. Transformation of tobacco wasconfirmed by measuring GUS activity (Jefferson et al., Embo J. (1987)6:3801-3807). A high transformation efficiency (80-100%) for transgenictobacco plants containing GUS gene activity was easily achieved usinghigh concentrations of A. tumefaciens LBA4404. Transformed tobacco wasincubated in MS medium comprising BAP and either IAA or 5-B-IAA topromote shoot regeneration. As shown in Table 5, both IAA and 5-B-IAAwere effective in stimulating shoot regeneration from transgenictobacco.

TABLE 5 Effect of 5-bromoindole-3-acetic acid on the regeneration ofshoots from Agrobacterium infected tobacco. Type % regeneration of shootof Ratio of BAP/auxin auxin 2.0 1.0 0.5 0.25 IAA 100% 80% 90% 90%5-B-IAA 80% 80% 90% 100%

Within a range of BAP/auxin concentration ratios of betweenapproximately 2.0 and 0.25, the efficiency of shoot regeneration fromtransgenic tobacco approached 100%, ranging from approximately 80-100%,as shown in Table 5.

b) Arabidopsis thaliana

For Arabidopsis thaliana, tissues of hypocotyl were removed from10-day-old seedlings and preincubated in MS complete medium containing0.5 mg/l 2,4-D and 0.5 mg/l kinetin for three days. The explants wereimmersed in 10⁹ cells/ml of Agrobacterium tumefaciens LBA4404 containingpBI121 for 20 min, and transferred to solid MS medium containing 500mg/l carbenicillin, 50 mg/l kanamycin, and various ratios of 5-B-IAA/2iPor IAA/2iP for callus and shoot formation. Arabidopsis explants wereincubated at 25° C., using an 18 h light/6 h dark cycle.

In transgenic A. thaliana, 5-B-IAA was a more effective auxin than IAAin promoting the regeneration of green callus.

TABLE 6 Effect of 5-bromoindole-3-acetic acid on the regeneration ofgreen callus from Agrobacterium infected Arabidopsis thalianaLandersburg leaves. Type % regeneration of green callus of Ratio ofCytokinin (2iP)/auxin auxin 2.0 1.0 0.5 0.25 IAA 30% 50% 90% 50% 5-B-IAA70% 100% 80% 70%

As shown in Table 6, the effective range for cytokinin/auxin ratios wasbetween approximately 2.0 and approximately 0.25. 100% green callusregeneration from transgenic A. thaliana was obtained at a 2iP/5-B-IAAratio of 1.0.

Example 5 The use of 5-B-IAA for Stimulating the Regeneration ofTransgenic Monocotyledonous Plants

The transformation of monocotyledonous plants is carried out accordingto art-known methods as described by Wu, “Methods for Transforming PlantCells,” in Plant Biotechnology (1989), Kung and Arntzen, Eds.,Butterworth Publishers, Stoneham, Mass. It is preferred thattransformation of monocots such as rice and wheat be performed by theparticle bombardment method as described in Wang et al. (1988) PlantMol. Biol. 11:433-439. The regeneration of transformed monocots isperformed according to known procedures (Vasil, Biotechnology (1988)5:387-402) as described in Example 4.

For example, rice (Oryza sativa) is transformed using the particlebombardment method of Wang et al. (supra) or the Agrobacterium-mediatingtechnique of Hiel et al. (1994) Plant Journal 6:271? or, alternatively,using the electroporation method as described by Dekeyser et al. (1990)Plant Cell 2:591-602. Regeneration of transformed rice is performedaccording to Abdullah et al. (1986) Bio/Technology 6:1087-1909 or,alternatively, according to Raineri et al. (1990) Bio/Technology8:33-38.

In a further example, maize is transformed and regenerated according tothe procedures of Rhodes et al. (1988) Bio/Technology 6:56-60 and (1988)Science 240:204-207.

In all cases, 5-B-IAA is used as the auxin to stimulate plant growth inaccordance with the invention. Where required, one or more additionalplant growth regulators may be added to the 5-B-IAA-comprising plantgrowth compositions.

All publications, patent applications and patents cited herein areincorporated by reference in the same extent as if each individualpublication, patent application or patent was specifically andindividually indicated to be incorporated by reference.

We claim:
 1. A method for affecting the growth of a plant locus, plantcell, plant tissue, immature plant seed, or mature plant seed whichcomprises applying to said plant locus, plant cell, plant tissue,immature plant seed, or mature plant seed, a growth-affecting, effectiveamount of a composition comprising 5-bromoindole-3-acetic acid, or asalt, an ester or an amide derivative thereof.
 2. The method of claim 1wherein said plant is a plant tissue culture.
 3. The method of claim 1wherein said plant is a monocotyledon.
 4. The method of claim 3 whereinsaid plant is Oryza sativa.
 5. The method of claim 1 wherein said plantis a dicotyledon.
 6. The method of claim 1 wherein said plant is atransgenic plant.
 7. The method of claim 6 wherein said plant is amonocotyledon.
 8. The method of claim 7 wherein said plant is Oryzasativa.
 9. The method of claim 6 wherein said plant is a docotyledon.10. The method of claim 9 wherein said plant is Arabidopsis thaliana.11. The method of claim 9 wherein said plant is Nicotiana tabacum.
 12. Amethod for enhancing the growth of a plant comprising the step ofgrowing said plant in the presence of a growth enhancing, effectiveamount of a composition comprising 5-bromoindole-3-acetic acid, or asalt, an ester or an amide derivative thereof, such that an improvementin a plant growth characteristic occurs in said plant in the presence ofsaid composition when compared to a corresponding plant in the absenceof said composition.
 13. The method of claim 12 wherein said compositionfurther comprises one or more plant growth hormone.
 14. The method ofclaim 12 wherein said composition comprises a cytokinin.
 15. The methodof claim 12 wherein said plant is a monocotyledon or a dicotyledon. 16.The method of claim 12 wherein said plant is a transgenic plant.